Tissue culture study of the medicinal plant leek (allium ampeloprasum L).

Persian shallot, also called leek (Allium ampeloprasum), is a monocotyledon plant of the lily family (Liliaceae). It belongs to the genus Allium, has a characteristic taste and morphological features, making it to be considered as one of the popular herbal medicine. This research was conducted with the purpose of obtaining optimal conditions for tissue culture of Persian shallot and comparing its active ingredient production in vitro versus in vivo. In this study, the auxin 2, 4-D and benzyl aminopurine- 6 (BAP) hormones, each at two concentrations (0.5 and 0.1 mg/ L) and Kin at 0.5 mg/ L were used in the format of a randomized complete block design in three replications. Results showed that the best culture media for callus formation for leaf and seed explants were the MS cultures with the hormonal compositions (0.5 mg/ L of 2, 4- D, 0.1 mg/ L of BAP) and (0.5 mg/ L of Kin and 0.1 mg/ L of 2, 4- D). Identification of the chemical composition of the essential oils, extracted either from leek callus or leaf was carried out using GC mass analysis. Twenty one compounds were detected in the GC mass spectra, seven of which constitutv about 51.5% of the total amount of compounds present in the essential oils were identified. Our data demonstrate that the leek essential oil constituents as well as callus formation can be affected by culture medium condition.


Extraction of essential oils
Extraction of the essential oils of Allium sp.

Chemical analysis
The essential oils were identified using the chromatography method by GC mass (model Hewelet 6890 with Detector 5975C). After extracting ther by the Clevenger, the essential oil was dried by dry sodium sulfate and then injected into the GC mass under the thermal scheduling program. Next, the constituents of each essential oil were determined.

Statistical analysis
The experimental data were evaluated by employing the SPSS software on the basis of Duncan's multiple range test at 5% probability level.

Leaf segments
The leaf segment explants and the seed samples started to swell gradually after they were cultured in the callus formation medium for two weeks, and started to produce calli after 3 weeks. In most experimental treatments, the majority of leaf explant samples and all seed samples turned into calli. Therefore, the weights of the calli formed in the treatments were compared in order to compare different treatments.
The comparison of means revealed that the effects of different hormonal compounds and their mutual effects on callus formation of leaf explants (Table 1) and seed samples were significant at 1% probability level.

Effects of the 2, 4-D and BAP on the callus
Comparison of the means of the data indicated that the hormonal treatments containing 0.5 mg/ L of 2, 4-D and 1 mg/ L of BAP (Table 2), and the hormonal treatments containing 0.1 mg/ L of 2, 4-D and 0.5 mg/ L of Kin (Table 3), had the greatest ability in forming calli. Therefore, these conditions were identified as the best treatments for callus formation.    Table 1. These results showed that the hormonal treatments had a significant effect on the percentage of callus formation.

GC-mass spectra
The percentage of the essential oils (w/w) were 0.3% from callus organ by the water extraction method. The peaks of the GC-mass of the essential oil of the plant leek obtained from leaf culture and the essential oil of its callus obtained by water extraction are presented in Figures 1 and 2 respectively.

Constituents of the essential oils of leaves and calli of leek
The active constituents in the leaves of leek accounting for 96.5% of compounds found in these leaves (shown in Table 4) were naphthalene, cycloisolongifolene, 3-methyl-4 isoprophylphenol, thymol, and caryphyllene, respectively.
Moreover, 30 g of wet callus of leek were extracted using the Clevenger apparatus, the extract was injected into the GC/ MS apparatus, and the compounds present in the extract were identified. Table 5 shows the contents of the active constituents present in the calli produced by culturing leek (which constitute 85.18 % of the total 100 % of the compounds found in the calli) .    These results show that the hormonal treatments have not had any significant effect on callus formation. The embryogenic callus cultures were friable, nodular and similar in appearance to those obtained leek (15)(16). The preferred callus type was often surrounded by soft watery callus; therefore, making sustained selection for the regeneration callus type is essential. Immature embryos have been successfully used to initiate embryogenic callus cultures for the major species inside the genus Allium (13,15,(17)(18).
The active constituents of essential oil according to table 5, were benzene, 7 Hcyclopenta, ethyl alpha-d-glucopyranoside, 2methylindene, 2H-1-benzopyran, 3.4-dihidro, respectively, and they were found in the largest quantities. Tables 4 and 5  The obtained results indicate that the leek essential oil constituents as well as callus formation can be affected by culture medium condition. Since the active ingredients of leek have many pharmacological properties, by controlling the culture medium ingredients, the quality and constituents of leek essential oil produced in vitro might be manipulated in a way to optimize the bioactive molecules production toward useful medicinal drugs.